Figure 2. Northern Blot Analysis of Novel miRNAs Identified by Computational Analysis.(A) Two novel C. elegans miRNAs,
mir-236 and
mir-228, show
dcr-1 dependent accumulation of the precursor miRNA form. Total RNA from wild-type and
dcr-1 (ok-247) mutant strains was subjected to Northern analysis using antisense probes against the mature form of
let-7 and the stem regions of the two C. elegans miRNA candidates. The mature (m) and precursor (p) forms of the miRNAs are indicated.(B) The expression of
mir-236 and
mir-228 is developmentally regulated. Northern blot of total RNA from wild-type C. elegans developmental stages probed as in (A) indicates a peak in expression at L1 larval stage. The mature (m) and precursor (p) forms of the miRNAs are indicated.(C) Expression of
mir-200b, a human homolog of
mir-236, and
mir-263, a fly homolog of
mir-228.
mir-200b was detected on a Northern blot of total RNAs from human tissues (top panel) and
mir-263 was detected on a Northern blot of total RNAs from fly developmental stages with antisense probes as described in (A).(D) Hairpin secondary structures for the sequence regions around which novel miRNAs are predicted to be encoded. The sequence boundaries of the precursor miRNAs remain to be determined. The stem regions to which the antisense probes were designed are indicated in red type; in the case of
mir-236 and
mir-263, predicted mature miRNA sequences were from the shortest probe which was positive by Northern blot. In cases where overlapping short probes were used, the full sequence including the nonoverlap region was used to designate the mature sequence in subsequent computational analyses.